Isolated molecules comprising epitopes containing sulfated moieties, antibodies to such epitopes, and uses thereof
A molecular and amino acid technology, applied in the field of tissue orientation and identification, which can solve the problems of difficult separation and display of monoclonal antibodies, high antibody immunogenicity, etc.
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Embodiment 1
[0602] Embodiment 1: the preparation of platelet
[0603] A citrate dextrose (ACD) concentrate of platelets was obtained from a blood bank, platelets were isolated and washed once in a buffer containing ACD and saline at a ratio of 1:7. Platelets were centrifuged at 800 g for 10 min after each wash and resuspended in Tyrode's solution (2 mM MgCl 2 , 137mM NaCl, 2.68mM KCl, 3mM NaH 2 PO 4 , 0.1% glucose, 5 mM Hepes and 0.35% albumin, pH 7.35) and count the number of cells.
[0604] Preparation of platelet-rich plasma
[0605] Blood was collected into tubes containing 3.8% sodium citrate. Platelet-rich plasma was prepared by centrifugation at 250 xg for 10 minutes.
Embodiment 2
[0606] Example 2: Platelet Aggregation
[0607] For platelet aggregation studies, platelet-rich plasma (PRP) and washed platelets were stirred at 500 rpm at 37°C in a whole blood Lumiaggregometer (Chronolog, Havertown, PA). The difference in light transmission through the platelet suspension and the suspended medium was defined as 100% agglutination. The effect of YI on platelet aggregation was assessed by adding different concentrations of Y1 prior to the addition of the agonist, and the effect was recorded for 4 minutes.
Embodiment 3
[0608] Example 3: Treatment of platelets with endoproteases
[0609] 3.1 Cutting of platelets by Mocarhagin
[0610] For mocarhagin digestion, dissolve 10 mM CaCl in TS buffer (0.01M Tris, 0.15M NaCl, pH 7.4) with 12 µg / mL mocarhagin (final concentration) at 22°C. 8 Washed platelets were treated for 1 hour and digestion was terminated by adding EDTA to 0.01M.
[0611] 3.2 Cleavage of glycocalicin by cathepsin G
[0612] 10 in TS buffer containing 1 mM calcium chloride 8 Washed platelets were incubated with 1.8 μg / ml cathepsin G (final concentration) for 4 hours at 22°C, and digestion was terminated by adding PMSF to 1 mM.
[0613] 3.3 O-sialoglycoprotein cleavage of glycocalicin
[0614] At 37 ° C, containing 0.2% albumin and protease inhibitors (10 μ M leupeptin, 0.24 mM PMSF) 10 6 Platelets were incubated with 0.14 mg / ml endo-sialoglycoproteinase (final concentration) for 45 minutes, and digestion was terminated by adding sample buffer and boiling. The sample buffer ...
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